2.1 Protozoal Related Disease
Protozoal parasites are single-celled organisms which live during some or all stages of their life cycle within organs, tissues and cells of multicellular, metazoan animals. As parasites, they obtain nutrients either from the host organism's food supply or from its cells and tissues. As eukaryotic unicellular organisms, the protozoal parasites are able to live both within animal cells and as free living extra-cellular parasites residing in the blood, lymph tissue or within the intestinal lumen.
As agents of infection, the protozoal parasites are fundamentally different than bacteria and viruses. Unlike bacteria and viruses, protozoa parasites are animals and share similar metabolism, respiration, and nutritional needs with their animal hosts. The similar metabolism of protozoa to mammalian metabolism renders most antibiotics and antiviral agents, selectively active against bacteria and viruses, respectively, ineffective for protozoal infection. Activity of compounds with antibacterial or antiviral activity against protozoal parasites would be atypical and unexpected. The lack of differences between protozoal metabolism and host cellular metabolism requires novel pharmacologic approaches to find therapeutic agents selective for eliminating protozoal organisms living within an animal host. Unlike bacteria and viruses, protozoa may assume different sexual forms and differentiate into a variety of maturational stages in various organs, presenting unique challenges for recognition by the host immune system. As genetically more complex organisms than bacteria and viruses, protozoa differentiate into forms which resist killing by known microbicides active against bacteria and viruses (Weir et al., 2002, Appl Environ Microbiol. 68(5):2576-9). Development of effective antiprotozoal therapeutics and stimulation of host immune responses against protozoal parasites therefore requires approaches different from those utilized in developing antibacterial, antiviral, and general immune potentiating agents.
The primary protozoal parasites causing disease in man include hemoflagellates of the class Trypanosomatidea, causing Leishmaniasis and Trypanosomiasis, and parasites of the phylum Apicomplexa, class Coccidea, causing malaria, toxoplasmosis, cryptosporidiosis, and bebesiosis. Species of Coccidea can infect humans, domestic animals and livestock, including poultry, lambs, calves, piglets, and rabbits. Protozoal parasitic diseases related to malaria include disease caused by parasites of the species Neospora. Neospora infections occur in dogs, cattle, sheep, goats and horses.
The majority of populations in developing countries are now at high risk of various protozoal infections including malaria, leishmaniasis, and trypanosomiasis. Together these protozoal diseases cause millions of preventable deaths every year. No preventive or therapeutic vaccines are yet available for these parasitic diseases. The market for drugs against such diseases is limited by poverty and the emergence of resistance to existing single agent chemotherapy. As used herein, chemotherapy refers to the use of chemical substances to treat disease. Due to the lack of protective immunity following infection, with or without chemotherapy, reinfection is a common phenomenon. Innovative and cost effective new drugs and combination therapies using new and old drug products are urgently needed. The development of broad-spectrum anti-parasitic agents able to be used in combination with existing chemotherapeutics is preferable to reduce the emergence of new resistance. An ideal anti-protozoal drug would target multiple protozoan parasites, be active by various routes of administration, reduce morbidity and mortality caused by such infections, not interfere with co-administered vaccines as they become available, and reduce the need for hospital-based treatment.
The important protozoal sources of infection addressed by the methods of treatment and compositions of the present invention are a subset of protozoal organisms within the biologic kingdom Protista. The protozoa relevant to the present invention are summarized in Table 1.
TABLE 1Selected field of parasites relevant to treatment methods and compositions using DIM-related indoles.PhylumClassOrderGenusSpeciesMicrosporidiaEnterocytozoon bieneusiChromistaBigyraBlastocystis hominisParabasaliaAxostylataParabasaleaTrichomonadidaTrichomonas vaginalisEuglenozoaSubphylum KinetoplastaTrypanosomatideaTrypanosomatidaLeishmaniaLeishmania speciesdonovani, infantum (chagasi), tropica,braziliensis, guyanensisTrypanosomaTrypanosoma speciesBrucei, gambiense, rhodesienseTrypanosoma cruziTrypanosoma rangeliApicomplexa (Sporozoa)CoccideaAdeleidaCryptosporidium baileyiCryptosporidium meleagridisCryptosporidium parvumCryptosporidium murisCyclospora cayetanensisIsospora belliCystoisosporaCystoisosporaEimeriidaToxoplasma gondiiHaemosporidaPlasmodiumPlasmodium falciparumPlasmodium malariaePlasmodium ovalePlasmodium vivaxBabesia gibsoniBabesia microtiConoidasidaEucoccidioridaNeosporaSarcosystis hominsSuihominis, lindemanni
2.2 Scope of Protozoal Parasitic Infections
Parasitic protozoa are responsible for a variety of human diseases transmitted by insect vectors, i.e., carriers, including malaria, leishmaniasis, and trypanosomiasis. Other protozoal parasites can be transmitted directly from other mammalian reservoirs or from person to person. Lacking vaccines, vector control and selective chemotherapy have been the only ways to reduce transmission and treat infected individuals, respectively. Because the immune system plays a crucial role in controlling protozoal infection, opportunistic infection with protozoal organisms is an increasing problem in infants, cancer patients, transplant recipients, and those co-infected with human immunodeficiency virus (HIV). Pregnancy also suppresses certain immune functions. New anti-protozal treatments are needed which are safer for mother and fetus during pregnancy, particularly for malaria, toxoplasmosis, and trichomonas infections. Vaccines are needed which overcome diminished immune responses and induce an adequate long term immune response. Vaccines can be used in conjunction with compatible chemotherapy to improve therapy of pre-existing chronic infection in endemic areas.
2.2.1 Malaria is an Uncontrolled Protozoal Disease
Malaria arises from infection with an Apicomplexan protozoan parasite known as Plasmodium. Only four species of the genus Plasmodium cause human malaria. P. vivax is the most common and fatal. P. ovale and P. malariae are less common and have intermediate severity. P. falciparum is the most virulent, responsible for high infant mortality, and associated with current drug resistance. The disease is transmitted to human beings through the bite of infected female Anopheles mosquitoes and by transfusion of infected blood.
Due to the emergence and spread of drug-resistant malaria parasites, pesticide-resistant malaria-transmitting mosquitoes, and population growth in endemic areas, malaria now causes approximately 500 million clinical cases per year. It is prevalent in children and pregnant women, causing about one million annual deaths in children under the age of five. Children growing up in rural and endemic areas are subject to more frequent malaria related illness and deaths than more resistant adults.
The most severe form of Plasmodium falciparum infection is cerebral malaria (CM). Cerebral malaria implies the presence of neurological features, especially impaired consciousness. Treatment of CM is limited to a few conventional anti-malarial drugs (quinine or artemisinins) and supportive care including parenteral fluids, blood exchange transfusion, osmotic diuretics and correction of hypoglycemia, acidosis and hypovolemia. The management of CM includes prompt administration of appropriate parenteral anti-malarial agents and early recognition and treatment of the complications. In children, the complications include severe anemia, seizures and raised intracranial pressure. In adults, renal failure and pulmonary edema are more common causes of death.
A number of drugs ranging from those of natural origin to synthetic ones have been developed for the treatment of malaria. Quinine and artemisinin are the commonly known drugs of natural origin, which are used for the treatment of malaria. A number of synthetic anti-malarial drugs such as chloroquine, mefloquine, primaquine, halofantrin, amodiaquine, proguanil, atovaquone, maloprim are known in the literature. Quinidine Gluconate, Quinine Sulfate, typically in combination with Doxycycline hyclate, Clindamycin, or Pyrimethamine-sufadoxine are also used for malaria. In chloroqine resistant strains, preferred oral therapy includes Mefloquine Hydrochloride and Atovaquone-proguanil hydrochloride combinations. In treatment of infections with P. vivax, P. malariae, P. ovale, and chloroquine sensitive P. falciparum, chloroquine phosphate and primaquine phosphate are used.
In recent years, drug resistant malaria has become one of the most serious problems in malaria control. Drug resistance necessitates the use of drugs which are more expensive and may have dangerous side effects. The emergence of resistance can be prevented by the use of combinations of drugs with different mechanisms of action. The use of drug combinations for all antimalarial treatment not only delays the onset of drug resistance, but also accelerates recovery and increases cure rates. A number of antimalarial combinations are already known in the field of malarial chemotherapy. The specific combinations in use, dosages, and relative merits of various combinations have been summarized (Kremsner et al., 2004, Lancet 364:285-94).
With the emergence of P. falciparum strains resistant to chloroquine and quinine, further alternative antimalarial chemotherapy is required. Due to frequent re-infection following complete or partial treatment, vaccine therapy promoting long term immunity to re-infection is needed. New chemotherapy will preferably clear the current infection and not interfere with co-administered vaccines as they become available. Preferred combinations of anti-malarials utilize drugs that overcome chloroquine resistance, have a good safety profile, and are well tolerated. Artemisinin, obtained from the plant Artemisia anua, and its derivatives are rapidly effective in severe malaria. Artemisinin compounds have been evaluated in several centers and are found to be effective, and safe (Miskra et al., 1995, Trans R Soc Trop Med Hyg 89:299-301).
In addition, the patent literature describes the combination of atovaquone and proguanil as a method for the treatment of malaria. See U.S. Pat. No. 5,998,449. The combination of fenozan with another anti-malarial agent selected from artemisinin, sodium artesunate, chloroquine, or mefloquine is described for the prophylactic and curative treatment of malaria. See U.S. Pat. No. 5,834,505. Synergistic combination kits using atemisinin derivatives, sulfadoxin and pyrimethamine for severe, multi-drug resistant malaria are described by Tipathi et al. in U.S. Patent Application Publication No. 2006/0141024 A1.
2.2.2 Trypanosomiasis Lacks Effective Chemotherapy for Early and Late Disease
African trypanosomiasis (sleeping sickness) is caused by a subspecies of the parasitic haemoflagellate, Trypanosoma brucei. The infection begins with the bite of an infected tsetse fly (Glossina spp.). Two forms of the disease are known, one caused by Trypanosoma brucei rhodesiense, endemic in Eastern and Southern Africa, and the other caused by T. b. gambiense, originally detected in West Africa, but also widespread in Central Africa. African Trypanosomiasis results in febrile, life-threatening illness in humans and also threatens livestock. T. brucei parasites rapidly invade the Central Nervous System (CNS) causing death within weeks if untreated. T. b. gambiense proliferates relatively slowly and can take several years before infecting the CNS system. There are four important drugs approved to treat these infections. Two of these, pentamidine and suramin, are used before the CNS involvement. The arsenic-based drug, melarsoprol is used in the case of infections established in the CNS. The fourth drug, eflornithine, is used against late stage infection caused by T. b. gambiense. This drug is ineffective against T. b. rhodesiense. Nifurtimox is another drug licensed for both American trypanosomiasis and melarsoprol-refractory late stage disease.
American trypanosomiasis or Chaga's disease is caused by Trypanosoma cruzi and effects millions of people in South and Central America, and Mexico. Untreated Chaga's disease causes decreased life expectancy due to parasitic cardiomyopathy and heart failure, megaesophagus, and megacolon. Blood-sucking triatomid bugs transmit the infection to young children and transplacental infection can occur with parasitemia during pregnancy. Nifurtimox and benznidazole are two drugs used for treatment of the acute disease, but are not known to be therapeutic for the chronic infection in older children and adults. In the absence of an effective vaccine, better agents are needed that can be taken prophylactially by at risk children. Following infection, additional agents are needed to be used in conjunction with nifurtimox and benznidazole to increase efficacy, permit lower doses of the current agents with reduced toxicity, and shorten the currently required duration of treatment.
2.2.3 Leishmaniasis Lacks Practical and Safe Chemotherapy
Human leishmaniasis comprises a heterogeneous spectrum of diseases. Three major forms are generally distinguished: cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis, of which the latter is potentially lethal. They are caused by various species of the protozoan parasite Leishmania and transmitted by female sandflies. The disease is currently estimated to affect some 12 million people in 88 countries. Worldwide, leishmania/HIV co-infection is now considered an emerging disease where about 50% of adult visceral leishmaniasis cases are related to co-existing HIV infection.
The current treatment for leishmaniasis involves administration of pentavalent antimony complexed to a carbohydrate in the form of sodium stibogluconate (Pentosam or Sb(V)) or meglumine antimony (Glucantine), which are the only established anti-leishmanial chemotherapeutic agents with a clearly favorable therapeutic index. The exact chemical structure and mode of action of pentavalent antimonials is still uncertain. Amphotericin B and Pentamidine are the second line of anti-leishmanial agents, but are reserved for non-responding infections due to potential toxicity. Since resistance to the antimony-based anti-Leishmanial drugs is emerging and treatment failures are common, new combination therapies are needed. Miltefosine is a recently introduced oral drug effective for visceral and cutaneous disease. The importance of this new oral agent extends to the treatment of dogs which serve as an important reservoir of the disease. The identification of additional, new and effective anti-leishmanial agents for oral administration would allow further treatment options, help prevent emerging resistance to Miltefosine and antimony-based drugs, and increase the chance for regional control of leishmaniasis. DIM has been shown to be a potent inhibitor of Leishmania donovani topoisomerase I (LdTOPILS) with an IC50 of 1.2 μM. See Roy A., et al., Biochemical Journal, 8 Oct. 2007, Immediate Publication Manuscript BJ 20071286 (not the final version).
2.2.4 Trichomonal Disease
Trichomonal infection, typically vulvo-vaginitis in women and urethritis in men, is sexually acquired and one of the most common protozoal parasite infections in humans. In the United States, it is estimated that more than 2 million women are infected each year. Trichomonas vaginitis causes vulvar itching and an odorous vaginal discharge. It is caused by Trichomonas vaginalis, a single-celled protozoan parasite not normally found in the flora of the genitourinary tract. Typically Trichomonal infection is treated with oral metronidazole which is FDA approved in various dosage regimens. Though efficacious, Metronidazole can exhibit serious dose-related side effects, particularly on the blood and on the central nervous system. Experiments show it to be mutagenic and carcinogenic. Recently, treatment failure and emerging resistance to metronidizole have been documented, indicating a need for more consistently effective therapies which will include combinations of drugs active against strains of T. vaginalis that may be resistant to metronidazole. Preferred treatments will include agents safe for pregnant women and allow lower doses of co-administered metronidazole.
2.2.5 Protozoal Disease in Immunocompromised Hosts
The risk of parasitic diseases is also present outside developing countries and often takes the form of chronic diarrheal disease in subjects with underlying immune deficiency. These infections can be caused by Isospora belli, and Cyclospora cayetanensis, both coccidian protozoa, where infection results in self-limited diarrhea in normal hosts and prolonged diarrhea in individuals with AIDS. Both infections respond to treatment with timethroprim-sufamethoxazole. Cryptosporidia are additional coccidian parasites that cause diarrhea in animal species and humans. Cryptosporidium parvum and C. Hominis account for most coccidial infections in humans. These organisms form oocytes, which when digested release sporozoites that invade host epithelial cells, penetrating the cell membrane but not the enterocyte cytoplasm. Nitazoxanide is the only drug approved for the treatment of cryptosporidiosis in the United States. The identification of additional effective anti-crytosporidial agents for oral use would allow additional treatment options for individuals with HIV infection who respond unpredictably to Nitazoxanide.
Toxoplasmosis, is a zoonotic infection by the obligate intracellular protozoan, Toxoplasma gondii. Toxoplasmosis is found throughout the world, including the United States. Cats and other feline species are the natural hosts for Toxoplasma gondii, however tissue cysts (bradyzoites) have been recovered from all mammalian species examined. Pregnant women and those with weak immune systems are particularly susceptible to the health risks resulting from Toxoplasma infection. Severe toxoplasmosis, particularly transplacental exposure, can result in damage to the brain, eyes, and other developing organs in utero. Currently available treatments for toxoplasmosis, which are the drugs trisulfa-pyrimdine, sulfadiazine and pyrimethamine, are not effective, and can be toxic to the host. Therefore, there is a need for therapeutic agents to treat toxoplasmosis that are more effective and less toxic than currently available treatment agents. No available agent is used to control Toxoplasmosis in cats.
2.3 Protozoal Cell Behavior Includes Apoptosis-Like Responses and Suppression of Apoptosis in Infected Host Cells
Apoptosis is the process of programmed cell death by which damaged cells are eliminated upon generation of unopposed death signals within the damaged cell. While apoptosis is primarily viewed as a biologic response of multicellular organisms providing a means of eliminating infected or transformed cells in the setting of viral and cancer-related disease, protozoal organisms have also been noted to exhibit programmed cell death behavior (Lee et al., 2002, Cell Death Differ. 9:53-64). When infecting host cells in mammals, protozoal parasites have also been noted to suppress host cell apoptosis. For example, activation of the Nuclear Factor Kappa B (NFκB) survival signaling pathway has been described following infection by Trypanosoma cruzi (Petersen et al., 2006, Infect Immun. 74:1580-7).
2.4 Natural Indole Compounds can Influence Apoptosis.
Cruciferous vegetables contain a family of plant protective compounds called glucosinolates which give rise to active compounds with indole rings exemplified by indole-3-carbinol (I3C). Oral ingestion of I3C results in the gastric conversion of I3C into at least twenty acid condensation products, many of which are bioavailable, the most prevalent of which include CTR (cyclic trimer; 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b′:7,8-b″]triindole), HI-IM (1-(3-hydroxymethyl)-indolyl-3-indolylmethane), DIM (diindolylmethane), ICZ (indolocarbazole) and LTr-1 (linear trimer; [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane) (Stresser et al., 1995, Drug Metabolism and Disposition 23:965-975). The fact that there are many non-DIM acid condensation products of I3C, produced in vivo at equal or greater levels as DIM, which can be responsible for I3C's activity, requires that biologic activities of individual condensation products like DIM be demonstrated directly.
As one of many products derived from I3C, DIM is also present in cruciferous plants following release of I3C. Once formed, DIM is stable in acid. In cell culture, isolated DIM has been shown to have apoptosis promoting effects in both estrogen-dependent and independent breast cancer cells (Hong et al., 2002, Biochem Pharmacol. 63:1085-97). In animals, orally administered DIM inhibits the growth of certain chemically induced forms of breast cancer (Chen et al., 1998, Carcinogenesis 19:1631-9). Recently, DIM has been shown to specifically induce apoptosis in Human Papilloma Virus (HPV) oncogene altered cervical cancer cell lines (Chen et al., 2001, J Nutr. 131:3294-3302). In further cell culture experiments, DIM has been shown to reduce activation of the NFκB signaling pathway in breast cancer cells (Rahman et al., 2005, Cancer Res. 65:364-71). Other non-DIM I3C condensation products were not tested. In vivo studies in mice suggest that expected effective plasma levels of DIM are not easily achieved in humans (Anderton et al., 2004, Drug Metab Dispos. 32:632-8).
In relation to its pro-apoptotic activity in tumor cells, DIM has also been shown to be estrogenic in breast cancer cells (Riby et al., 2000, Biochem. Pharmacol. 60:167-177) and in rainbow trout, a model of carcinogenesis relevant to cancer in humans (Shilling et al., 2001, Toxicology and Applied Pharmacology 170:191-200). Since estrogenic effects inhibit apoptosis, DIM may actually enhance estrogen related growth and survival of some cells. Based on the conflicting results of DIM's activity in cell culture studies and estrogenic activity in vivo, it is difficult to predict DIM's effects in vivo on protozoal disease processes. In addition, DIM has been shown to activate the Mitogen Activated Protein Kinase (MAPK) cell signaling pathway in cell culture (Leong et al., 2004, Mol Endocrinol. 18:291-302). Activated MAPK is associated with cancer promotion, cancer cell survival, and inhibition of apoptosis. These properties of DIM suggest that DIM would not be useful as a promoter of apoptosis in protozoal infection.
One approach, that has not been developed for protozoal parasitic disease, would be to selectively induce apoptosis in protozoal infected cells and tissues in order to cause the programmed death of parasites and of parasite infected cells. This would result in parasite clearance and increased apoptosis may support the development of protective host immunity.